PROJECT SUMMARY/ABSTRACT Maintenance of cellular homeostasis requires a fine-tuned balance of many biological processes. Protein homeostasis is particularly intricate, because it requires maintaining balance amongst biogenesis, folding, trafficking and degradation of all cellular proteins. A group of ubiquitous proteins, referred to collectively as molecular chaperones, play active roles in maintaining protein homeostasis by transiently binding to many different polypeptides. Of these, the Hsp70 chaperone/J-protein co-chaperone systems, which are present in all major cellular compartments, are the most versatile. They play key roles not only in general homeostasis networks of protein folding and degradation, but also in core biological processes, often by driving assembly and disassembly of multimeric complexes. The overarching goal of this proposal is to understand the attributes of Hsp70, and its J-protein co- chaperones, that drive their ability to carry out diverse biological roles. To do so we will use well-developed Hsp70/J-protein systems that are exceptionally amenable to both genetic and biochemical analysis. Knowledge generated using these systems will be portable to other organisms, as the Hsp70 and J-proteins being studied are highly conserved. It will also inform other Hsp70/J-protein systems that are less amenable to experimental analysis than those we employ as models. We are focusing on two major knowledge gaps. First, how Hsp70s interact with biologically relevant substrates. In the cell, polypeptides with substantial secondary and tertiary structure are Hsp70s' natural substrates. However, most work dissecting Hsp70's cycle of interaction with substrates has been done using peptide, because of the difficulty in working with partially folded proteins. Thus, understanding of how Hsp70s interact with these natural substrates is limited. Second, tethering to sites of action is a major means by which J-proteins drive Hsp70 function. But, that such tethering has evolved into complex and nuanced modes of functionality has only recently become evident. We will continue analysis of the eukaryotic ribosome associated J-protein/Hsp70 system. This system is fundamentally important. It not only plays a first-line role in de novo protein folding, it has been implicated in monitoring and modulating the translation process itself.